The discovery of various metabolites of Vitamin D.sub.3 has led to clinical studies assessing the significance of the presence or absence of these metabolites in various diseases and conditions. In order to properly evaluate the effect of the metabolite on the course of the disease, an appropriate detection and measurement system for the metabolites must be available. The normal level of 1.alpha.,25-dihydroxycholecalciferol (1.alpha.,25-diOH Vit D.sub.3) in man is 50-100 picograms. This low quantity of substance is extremely difficult to measure with an accuracy sufficient to see if there is an effect on the course of a disease or conditions due to the presence or absence of this material. Although certain diagnostic tests are available, they have been insufficciently sensitive and reproducible at the very low levels of substance being measured. A new reagent has been discovered which allows the sensitive, reproducible measurement of 1.alpha.,25-diOH Vit D.sub.3 and related compounds at very low concentrations.